The development of the ocular lens involves controlled proliferation an progressive differentiation of peripheral epithelial cells into terminally differentiated lens fibers. While knowledge regarding growth factors which regulate proliferation and differentiation in the lens is expanding, the elucidation of intracellular signaling pathways activated by receptor(s) for such growth factors represents a fundamental challenge in understanding how lens growth and differentiation are controlled. Recent studies have documented the expression of several proteins of the Ras GTPase superfamily in the lens, and demonstrated that inhibition of Rho GTPases impairs proliferation in lens epithelial cell cultures and leads to cataract formation in organ cultured lenses. These data support the hypothesis that the Rho GTPases are critical regulators of lens growth, development and function, and predict that Rho GTPases serve to integrate developmental responses to external growth cues. This application proposes to undertake a comprehensive investigation of the role(s) played by Rho GTPases during lens development and growth. First, the lens region-specific expression of the Rho GTPases (RhoA, B, and C), and their accessory proteins will be characterized, using immunodetection and ADP-ribosylation techniques. Lens epithelial cell culture systems will be utilized to assess the impact of deregulated Rho GTPase function on growth factor induced lens epithelial cell proliferation, apoptosis and cytoskeletal reorganization. This will be achieved by treating lens epithelial cells with recombinant C3-exoenzyme and by over-expression of functional mutants of RhoA. The role played by Rho-GTPases in vivo in lens development will be explored using transgenic mice with lens-specific expression of the C3 exoenzyme, a toxin which specifically inactivates Rho GTPases. These studies should provide detailed molecular insight into the role of Rho GTPase in particular, and growth factor responsive signaling pathways in general, in orchestrating proliferation and differentiation of the normal lens. Better understanding of signal transduction pathways regulating lens growth and development could provide more insights into etiology of cataract formation and novel approaches toward developing medical treatments for cataract.